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M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant

Moloney Murine Leukemia Virus Reverse Transcriptase, RNase H Minus (M-MLV RT [H–]), Point Mutant, is an RNA-dependent DNA polymerase that can be used in cDNA synthesis with long RNA templates (>5kb). The lack of RNase H activity is beneficial for this application, as RNase H can start to degrade templates when incubation times are long, as they may be when synthesizing long cDNAs. Although many researchers are successful in using M-MLV RT (H+) for analytical and some preparative cDNA applications, reverse transcriptases lacking RNase H activity provide another option to prepare l...

Moloney Murine Leukemia Virus Reverse Transcriptase, RNase H Minus (M-MLV RT [H–]), Point Mutant, is an RNA-dependent DNA polymerase that can be used in cDNA synthesis with long RNA templates (>5kb). The lack of RNase H activity is beneficial for this application, as RNase H can start to degrade templates when incubation times are long, as they may be when synthesizing long cDNAs. Although many researchers are successful in using M-MLV RT (H+) for analytical and some preparative cDNA applications, reverse transcriptases lacking RNase H activity provide another option to prepare long cDNAs and libraries containing a high percentage of full-length cDNA.

Features - Benefits

RNase H Minus: Provides optimal conditions for the preparation of full-length cDNA from long RNA templates.

Temperature Stability: Thermostability of this point mutant minimizes problems associated with secondary structure.

This product is available through the Promega Helix onsite stocking program in a –20°C Helix Freezer. The program offers numerous convenient solutions to meet your lab's needs. Helix Freezers are available in two sizes: 5.7 cubic ft. or 9.7 cubic ft.

This product is available through the Promega Helix onsite stocking program in a –20°C Helix Freezer. The program offers numerous convenient solutions to meet your lab's needs. Helix Freezers are available in two sizes: 5.7 cubic ft. or 9.7 cubic ft.

This product is available through the Promega Helix onsite stocking program in a –20°C Helix Freezer. The program offers numerous convenient solutions to meet your lab's needs. Helix Freezers are available in two sizes: 5.7 cubic ft. or 9.7 cubic ft.

cDNAs were synthesized from 1µg of total human RNA using M-MLV (H–), Point Mutant, (Panel A) or SuperScript® II (Panel B) reverse transcriptase at 37°C, 42°C and 50°C. GoTaq® Long PCR amplifications were performed on each transcription reaction using five primer sets, amplifying products from 9.4kb to 1.5kb. Ten microliters of the 9.4, 6.9, and 5.2kb PCR products and 1µl of the 3.2 and 1.5kb PCR products were separated on a 1% agarose gel. Lane M. BenchTop 1kb DNA Ladder (Cat.# G7541).

cDNAs were synthesized from 1µg of total human RNA using M-MLV (H–), Point Mutant, (Panel A) or SuperScript® II (Panel B) reverse transcriptase at 37°C, 42°C and 50°C. GoTaq® Long PCR amplifications were performed on each transcription reaction using five primer sets, amplifying products from 9.4kb to 1.5kb. Ten microliters of the 9.4, 6.9, and 5.2kb PCR products and 1µl of the 3.2 and 1.5kb PCR products were separated on a 1% agarose gel. Lane M. BenchTop 1kb DNA Ladder (Cat.# G7541).

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Figure 2. Sensitivity in a qPCR amplification.

Kanamycin control RNA was serially diluted tenfold (1 × 1010 to 1 × 101), spiked into 1ng of total human RNA and used as template in reverse transcription reactions with M-MLV (H–), Point Mutant, (Panel A) or SuperScript®II (Panel B) reverse transcriptase. Quantitative PCR amplification was performed on 5µl of the GoTaq® qPCR System with kanamycin control primers. Cq values are plotted versus molecules of RNA in the reverse transcription reactions. Cq values could not be determined at amounts less than 1 × 103 molecules. N=3.

Kanamycin control RNA was serially diluted tenfold (1 × 1010 to 1 × 101), spiked into 1ng of total human RNA and used as template in reverse transcription reactions with M-MLV (H–), Point Mutant, (Panel A) or SuperScript®II (Panel B) reverse transcriptase. Quantitative PCR amplification was performed on 5µl of the GoTaq® qPCR System with kanamycin control primers. Cq values are plotted versus molecules of RNA in the reverse transcription reactions. Cq values could not be determined at amounts less than 1 × 103 molecules. N=3.

/~/media/images/resources/figures/11100-11199/11146ma.jpg?la=en

Figure 3. Label incorporation.

Reverse transcription reactions were performed as described in the ChipShot™ Direct Labeling and Clean-UpSystem Technical Manual, #TM286, using the positive control transcriptase in the kit and M-MLV (H–), Point Mutant, or SuperScript® II reverse transcriptase. A no-reverse transcriptase reaction (–RT) was also performed as a negative control. Reactions were cleaned-up using the ChipShot™ Direct Labeling and Clean-Up System (Cat.# Z4100). The amount of cDNA generated and dye incorporated were determined using A260 and A550 values. N=2.

Reverse transcription reactions were performed as described in the ChipShot™ Direct Labeling and Clean-UpSystem Technical Manual, #TM286, using the positive control transcriptase in the kit and M-MLV (H–), Point Mutant, or SuperScript® II reverse transcriptase. A no-reverse transcriptase reaction (–RT) was also performed as a negative control. Reactions were cleaned-up using the ChipShot™ Direct Labeling and Clean-Up System (Cat.# Z4100). The amount of cDNA generated and dye incorporated were determined using A260 and A550 values. N=2.

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